Project Summary/Abstract G protein coupled receptors are essentially the molecular messengers of the cell, transducing signals from outside to inside. The signals are remarkably diverse, including a single photon, ion like calcium, neurotransmitters and hormones. Humans have more than 800 GPCRs for recognizing different signals, but only three types of transducers, G proteins, GPCR kinases (GRKs) and arrestins. GRKs phosphorylate GPCRs whereas G proteins and arrestins directly bind GPCRs, send the signal to downstream effectors and cause a cellular response. Comparing structures of GPCR bound to G protein and arrestin will provide valuable insights into the functional selectivity and guide the design of more potent drugs with better safety profiles. There are currently >300 structures of GPCR−G protein complex reported but only 8 published structures of GPCR−arrestin complex, which highlights the relative difficulty in obtaining suitable arrestin complexes for structural analysis. My lab focuses on the understudied GPCR−arrestin signaling pathway. We have developed a novel tool which stabilizes GPCR−arrestin complexes for cryo-electron microscopy studies and used that to visualize how the atypical chemokine receptor 3 engages arrestins in various ways at near-atomic resolution. For the next five years, we plan to expand the study to many important GPCR−arrestin pairs to understand the structural details to aid the design of selective interventions. This study will open the way to a detailed dissection of the mechanism of arrestin-mediated GPCR signaling but also to structure-based drug design.